Methotrexate exerts its primary cytotoxic effect by inhibiting which enzyme, leading to depletion of reduced folate cofactors?

Explanation

## Methotrexate: Mechanism of Action **Key Point:** Methotrexate is a folic acid analog that **irreversibly inhibits dihydrofolate reductase (DHFR)**, blocking the conversion of dihydrofolate to tetrahydrofolate (THF), the active form of folate. **High-Yield:** This inhibition depletes the **reduced folate pool**, which is essential for one-carbon transfer reactions required for: - Thymidylate synthesis (dTMP formation) - Purine synthesis (AICAR and FGAM transformylase steps) - Amino acid metabolism ### Mechanism Flowchart ```mermaid flowchart TD A[Methotrexate enters cell]:::action --> B[Polyglutamylation by FPGS]:::action B --> C[MTX-polyglutamate binds DHFR]:::action C --> D[Inhibition of DHFR]:::urgent D --> E[Dihydrofolate cannot → Tetrahydrofolate]:::urgent E --> F[Depletion of reduced folate pool]:::urgent F --> G[↓ dTMP synthesis]:::outcome F --> H[↓ Purine synthesis]:::outcome F --> I[↓ Amino acid metabolism]:::outcome G --> J[DNA synthesis blocked]:::urgent H --> J ``` ### Why DHFR, Not Thymidylate Synthase? | Enzyme | Inhibitor | Effect | Consequence | |--------|-----------|--------|-------------| | **DHFR** | **Methotrexate** | Blocks THF regeneration | Depletes entire folate pool; affects multiple pathways | | Thymidylate synthase | Fluorouracil (5-FU) | Blocks dTMP synthesis directly | Selective for DNA synthesis; less broad effect | **Clinical Pearl:** Methotrexate's effect is **not selective** for a single pathway — it starves the cell of all folate-dependent reactions, making it broadly cytotoxic. ### Polyglutamylation: The Activation Step 1. MTX enters via reduced folate carrier (RFC) 2. **Folylpolyglutamate synthetase (FPGS)** adds glutamate residues → MTX-polyglutamate 3. Polyglutamated form is **trapped inside the cell** and has **higher affinity for DHFR** 4. Resistance mechanism: ↓ FPGS activity or ↑ RFC mutations **Mnemonic:** **"MTX → DHFR → THF ↓ → dTMP ↓ & Purine ↓"** — one enzyme blocks the gateway to all folate-dependent synthesis. ### Rescue with Folinic Acid (Leucovorin) - Leucovorin is **5-formyl-THF**, the active form of folate - Bypasses DHFR block → restores THF pool - Used after high-dose MTX to prevent toxicity - **Timing:** Must be given 24–48 hours after MTX (after drug effect on tumor) [cite:KD Tripathi 8e Ch 65]